Ligand-specific dynamics of the progesterone receptor in living cells and during chromatin remodeling in vitro.

Progesterone receptor (PR), a member of the nuclear receptor superfamily, is a key regulator of several processes in reproductive function. We have studied the dynamics of the interaction of PR with a natural target promoter in living cells through the use of fluorescence recovery after photobleaching (FRAP) analysis and also have characterized the dynamics of the interaction of PR with the mouse mammary tumor virus (MMTV) promoter reconstituted into chromatin in vitro. In photobleaching experiments, PR in the presence of the agonist R5020 exhibits rapid exchange with the MMTV promoter in living cells. Two PR antagonists, RU486 and ZK98299, have opposite effects on receptor dynamics in vivo. In the presence of RU486, PR binds to the promoter and is exchanged more slowly than the agonist-activated receptor. In contrast, PR bound to ZK98299 is not localized to the promoter and exhibits higher mobility in the nucleoplasm than the agonist-bound receptor. Significantly, PR bound to R5020 or RU486 can recruit the SWI/SNF chromatin remodeling complex to the promoter, but PR activated with ZK98299 cannot. Furthermore, we found ligand-specific active displacement of PR from the MMTV promoter during chromatin remodeling in vitro and conclude that the interaction of PR with chromatin is highly dynamic both in vivo and in vitro. We propose that factor displacement during chromatin remodeling is an important component of receptor mobility and that ligand-specific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.

An estrogen receptor chimera senses ligands by nuclear translocation.

We have developed a new mammalian cell-based assay to screen for ligands of the estrogen receptor. A fluorescently tagged chimera between the glucocorticoid and the estrogen receptors, unlike the constitutively nuclear estrogen receptor, is cytoplasmic in the absence of hormone and translocates to the nucleus in response to estradiol. The chimera maintains specificity for estrogen receptor alpha ligands and does not show cross-reactivity with other steroids, providing a clean system for drug discovery. Natural and synthetic estrogen receptor alpha agonists as well as phytoestrogens effectively translocate the receptor to the nucleus in a dose-dependent manner. Antagonists of the estrogen receptor can also transmit the structural signals that result in receptor nuclear translocation. The potency and efficacy of high-affinity ligands can be evaluated in our system by measuring the nuclear translocation of the fluorescently labeled receptor in response to increasing ligand concentrations. The chimera is transcriptionally competent on transient and replicating templates, and is inhibited by estrogen receptor antagonists. Interestingly, the nucleoplasmic mobility of the chimera, determined by FRAP analysis, is faster than that of the wild type estrogen receptor, and the chimera is resistant to ICI immobilization. The translocation properties of this chimera can be utilized in high content screens for novel estrogen receptor modulators.

Subnuclear trafficking and gene targeting by steroid receptors.

Through the use of novel imaging techniques, we have observed direct steroid receptor binding to a tandem array of a hormone-responsive promoter in living cells. We found that the glucocorticoid receptor (GR) exchanges rapidly with regulatory elements in the continued presence of ligand. We have also reconstituted a GR-dependent nucleoprotein transition with chromatin assembled on promoter DNA, and we discovered that GR is actively displaced from the chromatin template during the chromatin remodeling process. Using high-intensity UV laser crosslinking, we have observed highly periodic interactions of GR with promoter chromatin. These periodic binding events are dependent on GR-directed hSWI/SNF remodeling of the template and require the presence of ATP. Both the in vitro and in vivo results are consistent with a dynamic model ("hit-and-run") in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is simultaneously lost from the template. We also find that receptor mobility in the nucleoplasm is strongly enhanced by molecular chaperones. These observations indicate that multiple mechanisms are involved in transient receptor interactions with nucleoplasmic targets.